METHOD OF ASSAYING DNA TOPOISOMERASES AND DNA BINDING PROTEINS USING HIGH THROUGHPUT SCREENING

摘要

A novel high throughput screening (HTS) technique to rapidly identify eukaryotic topoisomerase I active agents is presented. The method is based on genetic tagging of the topoisomerase I enzyme to directly immobilize the enzyme on a solid surface in a microtiter well format. For HTS operations, DNA is added to the wells and a fraction of the input plasmid is retained on the enzyme attached to the solid phase substratum. The retained DNA is detected by Picogreen fluorescence. Compounds that result in an increase in Picogreen staining represent potential topoisomerase interfacial poisons while those that reduce fluorescence report the presence of a catalytic inhibitor; therefore, the solid phase assay represents a 'bimodal' readout that reveals mechanisms of action. In addition to specific topoisomerase targeting drugs, the method also weakly detects other relevant anticancer agents, such as potent DNA alkylating and intercalating compounds; therefore, topoisomerase I HTS represents an excellent tool for searching and identifying novel genotoxic agents. This solid phase HTS is rapid, robust, economical and scalable for large library screens.